Visual signal transduction depends upon the GPCR rhodopsin and heterotrimeric guanine nucleotide binding proteins (G-proteins;G1 and G23). Structural characterization of opsin, rhodopsin, and G-proteins in multiple states has provided exceptional insight into the basic mechanisms of visual signaling. However, the molecular understanding of the G protein signaling cycle is far from complete. We are working to reveal the details of the complexes formed between signaling molecules. An important first step in this goal is to identify new methods to stabilize these normally transiently-formed complexes. Our first target will be stabilization of the rhodopsin- G123 signaling complex. We hypothesize that this complex will be most stable after light activation. This will provide a novel tool for future biochemical, structural and in vivo studies that will aim to identify how the mo- lecular architecture of the signaling complex catalyzes nucleotide release. We have designed two complemen- tary methods for the stabilization of this complex. In Aim 1: We will use cross-linking methods to stabilize the rhodopsin-transducin complex. We have already shown that a cross-link can be formed using bis(sulfosuccinimidyl) suberate (BS3) or its cleavable analog 3,3'- dithiobis-succinimidylpropionate (DTSSP). We are working to improve the efficiency of this reaction and to purify this complex away from unreacted rhodopsin. Cross-linked rhodopsin-transducin can be used for future biochemical studies including structural studies by electron microscopy or x-ray crystallography. In Aim 2: We will design site-directed mutants of the G1i subunit that have enhanced rhodopsin affinity and increase the duration of the meta II state. We have already identified two mutants with these properties, and will perform scanning mutagenesis on known regions of the rhodopsin-G1 subunit interface to identify further mutations that improve the stability of this normally transient complex. We anticipate that combination of several mutations will stabilize the rhodopsin-G123 complex. Successful stabilization of the complex using G1 subunit mutants can be used for future biochemical, structural, and in vivo studies. PUBLIC HEALTH RELEVANCE: We are working to define the mechanisms of visual signal transduction by investigating the transient complexes between G protein signaling molecules. This two year proposal develops a model system to stabilize the complex between the G protein coupled receptor rhodopsin and the G123 heterotrimer.